Hybridization chain reaction amplification for in situ imaging

ABSTRACT

The present invention relates to the use of fluorescently labeled nucleic acid probes to identify and image analytes in a biological sample. In the preferred embodiments, a probe is provided that comprises a target region able to specifically bind an analyte of interest and an initiator region that is able to initiate polymerization of nucleic acid monomers. After contacting a sample with the probe, labeled monomers are provided that form a tethered polymer. Triggered probes and self-quenching monomers can be used to provide active background suppression.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/371,346, filed Mar. 7, 2006, which is a non-provisional of U.S.patent application Ser. No. 60/659,499, filed Mar. 8, 2005. Thedisclosure of each of the priority applications is hereby incorporatedby reference in its entirety herein.

STATEMENT REGARDING FEDERALLY SPONSORED R&D

This invention was made with government support under grant nos. P01HD037105, R01 HD043897, and R01 HL078691 awarded by the NationalInstitutes of Health. The government has certain rights in theinvention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the use of hybridizationchain reaction for in situ imaging.

2. Description of the Related Art

Hybridization Chain Reaction (HCR) is a novel method for the triggeredchain of hybridization of nucleic acid molecules starting from stable,monomer hairpins or other more complicated nucleic acid structures. HCRis described in U.S. patent application Ser. No. 11/087,937, filed Mar.22, 2005, which is incorporated herein by reference. In the simplestversion of this process, stable monomer hairpins undergo a chainreaction of hybridization events to form a nicked helix when triggeredby a nucleic acid initiator strand. The fundamental principle behind HCRis that short loops are resistant to invasion by complementarysingle-stranded nucleic acids. This stability allows for the storage ofpotential energy in the form of loops; potential energy is released whena triggered conformational change allows the single-stranded bases inthe loops to hybridize with a complementary strand. HCR is described inco-pending provisional patent application Ser. No. 60/556,147 filed onMar. 25, 2004, incorporated herein by reference in its entirety, and oneembodiment is illustrated in FIG. 1. A more complex embodiment,quadratic HCR, is shown in FIG. 2.

In situ hybridization methods enable the detailed spatial mapping ofgenes and mRNAs in normal and pathological tissues, allowing the studyof gene expression and regulation in a morphological context from thesub-cellular to the organismal levels. Target nucleic acids areidentified via the hybridization of nucleic acid probes that facilitatesubsequent imaging by one of a number of methods. Radiolabeled probemolecules provide high sensitivity, but poor spatial resolution and thedisadvantage of working with biohazardous materials has motivated thedevelopment of several nonradioactive alternatives. In situ biologicalimaging of an analyte (including not only nucleic acids, but proteinsand other molecules and compounds) or multiple analytes can beaccomplished by a variety of methods; however, such techniques havelimitations, particularly as the number of analytes increases.

Fluorescence in situ hybridization (FISH) methods enable the detailedmapping of transcriptionally active genes from sub-cellular toorganismal levels.(Lawrence et al. Cell, 57:493-502, 1989; Kislauskis etal. The Journal of Cell Biology, 123(1):165 172, 1993; Wilkie et al.Current Biology, 9: 1263-1266, 1999; Levsky, et al. Science297:836-840,2002; D. Kosman, et al. Science, 305:846, 2004.)Fluorescently-labeled nucleic acid probes are amenable to multiplexing(Levsky, 2002) but provide low sensitivity due to the small number ofdyes per probe (Kosman, 2004). Immunological methods employ antibodiesto bind haptenated nucleic acid probes which are then detected usingfluorescently-labeled secondary antibodies (Kosman, 2004; Hughes et al.BioTechniques, 24(4):530-532, 1998). Some amplification can be achievedby introducing additional layers of labeled antibodies, (P. T. Macechko,et al. J Histochem Cytochem, 45(3):359-363, 1997) but the sensitivity isinsufficient for imaging low-abundance mRNAs. Spatially localized signalamplification can be achieved using horseradish peroxidase-labeledantibodies (Wilkie, 1999; Kosman, 2004) or probes (M.P.C. van de Corputet al. J Histochem Cytochem, 46(11):1249-1259, 1998) to catalyze thebinding of fluorescent tyramides in the vicinity of the probe. Thisapproach significantly enhances sensitivity, but serial multiplexingresults in sample degradation. Similar sensitivities have been achievedusing in situ PCR, but the method is more cumbersome and the results areless reproducible. All of the above methods suffer from enhancedbackground signal due to the nonspecific binding of nucleic acid probesprior to fluorescent labeling or amplification.

Another technique for amplifying a signal from a hybridization event isthe branched DNA (bDNA) approach, in which a pre-amplifier strandhybridizes to a portion of the probe, which in turn serves as anucleation site for the hybridization of a fixed number ofmultiply-labeled amplifier strands. (Schweitzer et al. Curr OpinBiotechnol, 12:21-27, 2001.; Qian et al. Diagnostic Molecular Pathology,12(1):1-13, 2003; Collinset al. Nucleic Acids Res, 25(15):2979-2984,1997.; Bushnell, et al. Bioinformatics, 15(5):348-355, 1999; Player, etal. J Histochem Cytochem, 49(5):603-611, 2001.)

SUMMARY OF THE INVENTION

Methods and compositions for detecting one or more analytes within abiological sample (in situ) using HCR are provided. The advantages ofHCR for in situ imaging include, without limitation, the ability torapidly amplify a signal based on a small amount of analyte present andthe ability to image a diversity of analytes in the same sample.

In one aspect of the invention, methods are provided for detecting ananalyte in a biological sample. Preferably, the sample is contacted witha probe comprising a target region and an initiation region. The targetregion is able to specifically bind to the analyte of interest, whilethe initiation region is able to initiate the polymerization of labelednucleic acid monomers. Thus, the sample is contacted with a firstmetastable monomer comprising an initiator region that is complementaryto the initiation region of the probe and a second metastable monomercomprising a region complementary to a portion of the first monomer. Oneor both of the monomers is preferably labeled with a fluorescent dye.They may also be labeled with a fluorescence quencher such that prior topolymerization the fluorescence is quenched. A fluorescent signal isthus generated upon formation of a polymer and background is reduced.

The analyte to be detected is not limited in any way and may be, forexample, a nucleic acid such as mRNA or a gene of interest, or apolypeptide. In some preferred embodiments the analyte is a nucleic acidand the target region of the probe is complementary to at least aportion of the analyte.

In some embodiments, a triggered probe is utilized, such that theinitiation region is only made available to interact with the monomerswhen the probe is bound to the analyte of interest. For example, in someembodiments the probe undergoes a conformational change upon binding ofthe target region to the analyte such that the initiation region isavailable to stimulate polymerization. In this way, non-specificpolymerization resulting from non-specific probe binding is reduced.

The in situ HCR reactions can be multiplexed to identify the presence ofmultiple analytes of interest simultaneously.

In another aspect, methods of in situ imaging are provided in which abiological sample is contacted with a probe comprising a target regioncapable of specifically binding to an analyte of interest and aninitiator region, such that the probe binds to the analyte of interest.The sample is then contacted with at least two fluorescently labeledmonomers, whereby the initiator region of the bound probe hybridizes toat least one of the monomers. As a result, the monomers form afluorescently labeled polymer tethered to the analyte via the probe. Thefluorescently labeled polymer can then be visualized.

In a further aspect, kits are provided for the in situ detection of ananalyte of interest, preferably a nucleic acid. The kits preferablycomprise a first metastable nucleic acid monomer comprising an initiatorcomplement region and a fluorescent label and a second metastablenucleic acid monomer comprising a propagation region that issubstantially complementary to a portion of the first nucleic acid. Aprobe is included comprising a target region that is complementary to atleast a portion of the nucleic acid to be detected and an initiatorstrand that is complementary to the initiator complement region of thefirst monomer. The first and second monomers are preferably hairpinmonomers. In some embodiments the kits comprise one or more additionalmonomers and may comprise one or more additional probes, for identifyingmultiple analytes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically illustrates one embodiment of an HCR system. Eachletter represents a segment of nucleic acids. Letters marked with a *are complementary to the corresponding unmarked letter.

FIG. 1A shows two hairpins, labeled H1 and H2, that are metastable inthe absence of initiator I. The hairpins comprise sticky ends ‘a’ and‘c*’, respectively. Potential energy is stored in the hairpin loops.

FIG. 1B shows how a single initiator strand ‘I’ can nucleate or bind tothe sticky end of H1 and displace one arm to open the hairpin. Thisfrees up the bases that were trapped in the hairpin, allowing them toperform a similar displacement reaction on H2.

As illustrated in FIG. 1C, the newly exposed c region of H1 nucleates atthe sticky end of H2 and opens the hairpin to expose a region on H2 (a*)that is identical in sequence to the initiator I. As a result, each copyof I can propagate a chain reaction of hybridization events betweenalternating H1 and H2 hairpins to form a nicked double helix, therebyamplifying the signal of initiator binding. The process can continueuntil the monomers (H1 and H2) are exhausted. At each step, energy isgained from the hybridization of ‘a’ or ‘c’ The reactions diagrammed inFIG. 1 have been successfully carried out and are summarized in FIG. 1D.

FIG. 1D illustrates the results of an HCR reaction and the effect ofinitiator concentration on amplification. Lanes 2-7: Six differentconcentrations of initiator were used (0.00, 10.00, 3.20, 1.00, 0.32 and0.10 μM, respectively) in a 1 μM mixture of H1 and H2. Lanes 1 and 8were DNA markers with 100-bp and 500-bp increments respectively.

FIG. 1E illustrates HCR kinetics monitored by substituting afluorescently labeled base in the sticky end of an HCR monomer. Here2-aminopurine (2AP) was substituted for A (base 3) in the sticky end ofH1. The hairpin monomers H1 and H2 did not hybridize prior to triggeringby initiator ((H1^(2AP)+1.2× H2 for 24 hours+0.5× initiator), red). Thesame quenched baseline was achieved without HCR by adding excessinitiator to H1^(2AP) the absence of H2 (H1^(2AP)+4.0× initiator,green). Addition of insufficient initiator to H1^(2AP) only partialquenching (H12AP+0.5× initiator (blue), demonstrating that HCR, and notinitiator alone, was responsible for exhausting the supply of H12APmonomer.

FIG. 2 illustrates an embodiment in which HCR utilizing two monomerpairs produces quadratic signal amplification. FIG. 2A illustrates twohairpin monomers Q1 and Q2 that are metastable in the absence ofinitiator IQ. As shown in FIG. 2B, binding of IQ leads to a stranddisplacement interaction that exposes sticky end fe*b*. This singlestranded region then nucleates at the sticky end of Q2, and a subsequentbranch migration exposes segments cb*d* and e* as shown in FIG. 2C. Thed*e* region initiates the next Q1 molecule, leading to amplification inone direction, while the exposed cb* region initiates a second HCRreaction involving monomers H1 and H2 (FIG. 1). As illustrated in FIG.2D, the resulting branched polymer has a Q1/Q2 main chain with H1/H2side chains branching off at each Q2 segment.

FIG. 3 illustrates a pair of monomers (E1 and E2) that can be used incombination with at least one other pairs of monomers to achieveexponential amplification by HCR. In the presence of cb*, E1 and E2 forma linear chain that includes periodic single stranded d*e* regions. Theinitiator sequence for E1/E2 matches the periodic single stranded regionproduced by Q1/Q2 and vice versa. Consequently, a mixture of Q1, Q2, E1and E2 plus either initiator (cb* or d*e*) will lead to the formation ofa structure in which each branch of the polymer is itself a branchedpolymer. Sustained growth will ultimately decrease to cubicamplification.

FIGS. 4A-E illustrate another embodiment for HCR with exponentialgrowth. Eight different strands are used in this embodiment. Strand one(1) is the ‘hub’ of the system and has an exposed, single-strandedregion joining two hairpins (FIG. 4A). When the initiator (2) binds, itcreates a long helix with one sticky end on each side. The two stickyends generated by the initiated ‘hub’ bind with strands (3) and (6),respectively (FIG. 4B and C). Next, auxiliary strands (4) and (7) bindto previously protected bulge loops (FIGS. 4D and E), and expose twohairpin regions. These hairpins then bind to strands (5) and (8),respectively, to generate sticky ends similar to the initiator molecule(2). Thus, each initiator produces two new initiators, leading toexponential growth. As a side note, two subsets of strands (1,2,3,4,5)and (1,2,6,7,8) produce linear systems in the absence of the otherstrands.

FIG. 5A illustrates an aptamer HCR trigger mechanism for the detectionof ATP. Binding of the DNA aptamer to ATP induces a conformation changethat exposes a sticky end.

FIG. 5B shows an agarose gel demonstrating ATP detection via HCR.

FIG. 6 illustrates a self-complementary hairpin with an interior loopthat is a double helix (dotted lines). DNA hairpin can also exist as adimer with an interior loop. One possible concern is that the interiorloops may be easier to invade than the corresponding hairpins. Toprevent this side reaction, self-complementary hairpins would convertthe interior loop to a simple double helix (dotted lines). However, thisadded precaution may not be necessary.

FIG. 7 is a picture of a gel showing independent operation of two HCRsystems in the same solution. System 1 comprised hairpins H1A and H1Band initiator I1 . System 2 comprised hairpins H2A and H2B and initiator12. Hairpins H1B and H2B were 5′-labeled with fluorophores FAM (redsignal) and Cy3 (green signal), respectively. All reactions containedall four hairpin species at 0.5 μM. Lane 1: No reaction was seen in theabsence of initiators. Lanes 2 and 3: specific detection of I1 (1× and0.25×, respectively). Lanes 4 and 5: specific detection of I2 (1× and0.25×, respectively). Lanes 6 and 7: simultaneous detection of I1 and I2(1× and 0.25×, respectively).

FIG. 8 is a schematic illustration of HCR probes for in situ HCRamplification. A standard probe illustrated in FIG. 8A leaves the HCRinitiator exposed at all times. A triggered probe (FIG. 8B) protects theinitiator until specific binding of the probe to the target mRNA exposesthe initiator. An alternative triggered probe (FIG. 8C) reduces thesequence dependence of the amplifier hairpins on the target mRNA(sequence b* is now independent of the target sequence).

FIG. 9 illustrates an HCR antibody probe.

FIG. 10 schematically illustrates fluorescent labeling schemes for insitu HCR amplification. As shown in FIGS. 10A and C fluorescentend-labeling leads to fluorophores at ≈8 nm spacing in the assembledamplification polymers. Self-quenching hairpins (FIGS. 10B and D) withfluorophore/quencher pairs reduce the background signal further for invivo applications, particularly where unused hairpins cannot be washedout of the sample. The fluorophore/quencher distance increases from nmto nm after HCR amplification polymers have assembled, leading toincreased fluorescence.

FIG. 11 illustrates a scheme for quantitative nonlinear HCRamplification. FIG. 11A illustrates the HCR amplification components.Strand I corresponds to the initiator sequence that is exposed when atriggered probe binds specifically to its target. FIG. 11B illustrates apartially assembled branched amplification polymer. FIG. 11C shows abinary branching tree for an amplification polymer with fourgenerations.

DETAILED DESCRIPTION

Hybridization Chain Reaction (HCR) is a method for the triggeredhybridization of nucleic acid molecules starting from metastable monomerhairpins or other metastable nucleic acid structures. See, for example,Dirks, R. and Pierce, N. Proc. Natl. Acad. Sci. USA 101(43): 15275-15278(2004), and U.S. patent application Ser. No. 11/087,937, filed Mar. 22,2005, each of which is incorporated herein by reference in its entirety.HCR does not require any enzymes and can operate isothermally.

In one embodiment of HCR, two or more metastable monomer hairpins areused. The hairpins preferably comprise loops that are protected by longstems. The loops are thus resistant to invasion by complementarysingle-stranded nucleic acids. This stability allows for the storage ofpotential energy in the loops. Potential energy is released when atriggered conformational change allows the single-stranded bases in theloops to hybridize with a complementary strand, preferably in a secondhairpin monomer.

Each monomer is caught in a kinetic trap, preventing the system fromrapidly equilibrating. That is, pairs of monomers are unable tohybridize with each other in the absence of an initiator. Introductionof an initiator strand causes the monomers to undergo a chain reactionof hybridization events to form a nicked helix (see FIGS. 1A-C). HCR canbe used, for example, to detect the presence of an analyte of interestin a sample. This and other applications are discussed in more detailbelow.

Methods and compositions for detecting one or more analytes within abiological sample (in situ) using HCR are provided. The advantages ofHCR for in situ imaging include, without limitation, the ability torapidly amplify a signal based on a small amount of analyte present andthe ability to image a diversity of analytes in the same sample.

As described herein the use of HCR for in situ detection and imagingprovides a number of advantages. Specificity can be achieved by usingtriggered probes that protect the initiators until the probes bindspecifically to targets. Self-quenching HCR monomers can be labeled withfluorophore/quencher pairs that become separated during self-assemblyinto tethered amplification polymers. This active background suppressionis particularly useful for in vivo applications where unusedamplification components cannot be washed away before imaging.Versatility can be achieved by selecting structure-switching aptamers(Ellinton et al. Nature 346:818-822, 1990 and Tuerk et al. Science249:505-510, 1990) that generalize the triggered probe concept to thedetection of proteins and small molecules. Small probe and amplificationmonomers, preferably with maximum dimensions of 8-16 nm facilitatesample penetration. Isothermal conditions are ideal for HCRamplification, avoiding damage to the morphology of fixed samples ortheir components and facilitating in vivo imaging. Multiplexing followsnaturally from the use of independent HCR amplifiers that operatesimultaneously, for example using spectrally distinct fluorophores toencode unique combinatorial signatures directly into the structure ofeach HCR product. Sensitive quantitative amplification can be acheivedusing nonlinear HCR mechanisms that offer exponential growth intotethered polymers of a prescribed finite size. Finally, biocompatibilityfor in vivo applications follows from the use of nucleic acid amplifiercomponents.

Definitions

“Nucleic Acids” as used herein means oligomers of DNA or RNA. Nucleicacids may also include analogs of DNA or RNA having modifications toeither the bases or the backbone. For example, nucleic acid, as usedherein, includes the use of peptide nucleic acids (PNA). The term“nucleic acids” also includes chimeric molecules.

The term “sticky end” refers to a nucleic acid sequence that isavailable to hybridize with a complementary nucleic acid sequence. Thesecondary structure of the “sticky end” is such that the sticky end isavailable to hybridize with a complementary nucleic acid under theappropriate reaction conditions without undergoing a conformationalchange. Typically the sticky end is a single stranded nucleic acid.

“Monomers” are individual nucleic acid oligomers. Typically, at leasttwo monomers are used in hybridization chain reactions, although three,four, five, six or more monomers may be used. In some embodiments morethan two monomers are utilized, such as in the HCR systems displayingquadratic and exponential growth discussed below. Typically each monomercomprises at least one region that is complementary to at least oneother monomer being used for the HCR reaction.

A first monomer in a monomer pair preferably comprises an initiatorcomplement region that is complementary to a portion of an initiatormolecule. The initiator complement region is preferably a sticky end.Binding of the initiator to the initiator complement region begins anHCR reaction.

In addition, the second monomer preferably comprises a propagationregion that is able to hybridize to the initiator complement region ofanother monomer, preferably another copy of the first monomer, tocontinue the HCR reaction begun by the initiator. The propagation regionmay be, for example, the loop region of a hairpin monomer as describedbelow. In one embodiment the propagation region on the second monomer isidentical to the portion of the initiator that is complementary to theinitiator complement region of the first monomer.

The propagation region on the second monomer is preferably onlyavailable to interact with the initiator complement region of the firstmonomer when an HCR reaction has been started by the initiator. That is,the propagation region becomes available to hybridize to the initiatorcomplement region of another monomer when one copy of the first monomerhas already hybridized to a second monomer, as discussed in more detailbelow.

Preferred monomers are “metastable.” That is, in the absence of aninitiator they are kinetically disfavored from associating with othermonomers comprising complementary regions. “HCR” monomers are monomersthat are able to assemble upon exposure to an initiator nucleic acid toform a polymer.

As used herein, “polymerization” refers to the association of two ormore monomers to form a polymer. The “polymer” may comprise covalentbonds, non-covalent bonds or both. For example, in some embodiments twospecies of monomers are able to hybridize in an alternating pattern toform a polymer comprising a nicked double helix. The polymers are alsoreferred to herein as “HCR products.”

An “initiator” is a molecule that is able to initiate the polymerizationof monomers. Preferred initiators comprise a nucleic acid region that iscomplementary to the initiator complement region of an HCR monomer.

Monomers

Two or more distinct species of nucleic acid monomers are preferablyutilized in an HCR reaction. Each monomer species typically comprises atleast one region that is complementary to a portion of another monomerspecies. However, the monomers are designed such that they arekinetically trapped and the system is unable to equilibrate in theabsence of an initiator molecule that can disrupt the secondarystructure of one of the monomers. Thus, the monomers are unable topolymerize in the absence of the initiator. Introduction of an initiatorspecies triggers a chain reaction of alternating kinetic escapes by thetwo or more monomer species resulting in formation of a polymer. In theexamples below, the two hairpin monomers polymerize in the presence ofan initiator to form a nicked, double helix.

In a preferred embodiment, two or more monomer species are employed thathave a hairpin structure. The hairpin monomers preferably comprise loopsprotected by long stems. In other embodiments, monomers with a differentsecondary structure are provided. However, the secondary structure ispreferably such that the monomers are metastable under the reactionconditions in the absence of an initiator nucleic acid. In the presenceof an initiator, the secondary structure of a first monomer changes suchthat it is able to hybridize to a sticky end of a second monomerspecies. This in turn leads to a change in the secondary structure ofthe second monomer, which is then able to hybridize to another firstmonomer and continue the process. In this way, once a single copy of thefirst monomer interacts with a single copy of the initiator, a chainreaction is produced such that the monomers are able to assemble into apolymer comprising alternating monomer species.

A number of criteria can be used to design the monomers to achieve thedesired properties. These include, for example and without limitation,sequence symmetry minimization, the probability of adopting the targetsecondary structure at equilibrium, the average number of incorrectnucleotides at equilibrium relative to the target structure, andhybridization kinetics.

Monomers can be synthesized using standard methods, includingcommercially available nucleic acid synthesizers or obtained fromcommercial sources such as Integrated DNA Technologies (Coralville,Iowa).

In some embodiments, monomers are derivitized with a compound ormolecule to increase the molecular weight of the polymer resulting fromHCR. Preferably they are derivitized at a location that does notinterfere with their ability to hybridize. In other embodiments monomerscomprise a fluorophore or colorimetric compound that allows theresulting polymers to be visualized.

In preferred embodiments, at least two hairpin monomers are utilized asillustrated in FIG. 1A. The monomers each preferably comprise a stickyend (a and c*, respectively), a first complementary segment (b and b *,respectively), a loop segment (c and a*, respectively), and a secondcomplementary segment (b and b*, respectively). The first and secondcomplementary segments are also referred to as “stems” and together forma duplex region.

The first monomer (H1) preferably comprises a sticky end a that iscomplementary to a first nucleic acid portion a* of an initiator (I;FIG. 1B). This sticky end is referred to herein as the “initiatorcomplement region.” The initiator may be, for example, an analyte ofinterest, or a nucleic acid that is able to contact the first monomeronly in the presence of an analyte of interest, as discussed in moredetail below.

The second monomer (H2) preferably comprises a sticky end c* that iscomplementary to a portion of the first monomer that becomes accessibleupon initiator binding. Preferably the sticky end c* is complementary tothe loop segment c of the first monomer (FIG. 1A). The loop segment c ofthe first monomer is preferably not available to hybridize with stickyend c* of the second monomer in the absence of initiator.

The first and second complementary segments (b and b*) in the first andsecond monomers are typically substantially identical. That is, thefirst complementary segment b of the first monomer (H1) is able tohybridize to the second complementary segment b* of the second monomer(H2).

The first complementary segment of each monomer is also able tohybridize to the second complementary segment of the same monomer toform the hairpin structure. For example, as shown in FIG. 1A, the firstmonomer (H1) comprises a first complementary segment b that is able tohybridize to the second complementary segment b* . In the absence of aninitiator, the first and second complementary segments of each monomerare generally hybridized to form a duplex region of the metastablemonomer.

Preferably, the first complementary segment b of the first monomer isalso complementary to a portion b* of the initiator, such that uponhybridization of the initiator region a* to the sticky end a (theinitiator complement region) of the first monomer H1, one arm of thehairpin structure is displaced. This opens the hairpin and allowsbinding of the first complementary segment b to the second portion b* ofthe initiator strand (FIG. 1B).

The loop segment c of the first monomer is also exposed by the openingof the hairpin and is able to bind to the sticky end c* of the secondmonomer H2, as illustrated in FIG. 1C. This opens the second monomerhairpin H2 and the second complementary segment b* of the first monomeris able to hybridize to the first complementary segment b of the secondmonomer H2.

This leaves the loop region a* and first complementary region b* of thesecond monomer H2 exposed (FIG. 1C). The sticky end a of another firstmonomer (H1) species is able to bind to the exposed loop region a* ofthe second monomer H2, thus opening the H1 hairpin and continuing theprocess described above. Because the loop region a of the second monomeracts as an initiator on a second H1 monomer and allows the process tocontinue in the absence of further initiator, it is referred to as thepropagation region.

At each step, energy is gained from the hybridization of the sticky endof the monomer. The result is a nicked, double helix polymer comprisingalternating H1 and H2 fragments. This process preferably continues in achain reaction until all of one or both of the monomer species is usedup, or the reaction is stopped by some other mechanism. If desired, thenicks in the nucleic acid polymer structures that result from HCR can byligated (for example, using T4 DNA ligase).

Because of the self-propagating nature of the reaction, each copy of theinitiator species can begin the chain reaction. Further, as long asthere is a fixed supply of monomers the average molecular weight of theresulting polymers is inversely related to the initiator concentration,as can be seen in FIG. 1D.

The length of the loop, stem and sticky ends of the monomers can beadjusted, for example to ensure kinetic stability in particular reactionconditions and to adjust the rate of polymerization in the presence ofinitiator. In one preferred embodiment the length of the sticky ends isthe same as the length of the loops. In other embodiments the stickyends are longer or shorter than the loops. However, if the loops arelonger than the sticky ends, the loops preferably comprise a region thatis complementary to the sticky end of a monomer.

In some preferred embodiments the length of the loops is short relativeto the stems. For example, the stems may be two or three times as longas the loops.

The loop regions are preferably between about 1 and about 100nucleotides, more preferably between about 3 and about 30 nucleotidesand even more preferably between about 4 and about 7 nucleotides. In oneembodiment the loops and sticky ends of a pair of hairpin monomers areabout 6 nucleotides in length and the stems are about 18 nucleotideslong.

Other refinements to the system stabilize the monomer hairpins to helpprevent HCR in the absence of an initiator. This can be achieved, forexample, via super-stable hairpin loop sequences (Nakano et al.Biochemistry 41:14281-14292 (2002)), with ostensible structural featuresthat could further inhibit direct hybridization to the hairpin. In otherembodiments hairpin loops are made to be self-complementary at theirends. This self-complementarity “pinches” the hairpin loops, making themshorter. However, if the reactive sticky ends of each monomer arecomplementary to the loop regions on the opposite monomer, as describedabove, they will have a slight propensity to close up, thereby slowingdown the reaction. This feature can be utilized if a slower reaction isdesired Completely self-complementary hairpins can also be used, forexample if the monomer hairpins are forming dimers with interior loopsthat are more easily invaded than their hairpin counterparts. FIG. 6illustrates a self-complementary hairpin with an interior loop that is adouble helix.

Reaction conditions are preferably selected such that hybridization isable to occur, both between the initiator and the sticky end of a firstmonomer, and between the complementary regions of the monomersthemselves. The reaction temperature does not need to be changed tofacilitate the hybridization chain reaction. That is, the HCR reactionsare isothermic. They also do not require the presence of any enzymes.

Variations

There are many possible variations to HCR that may improve its speed,stability and ability to amplify chemical signals. The systemillustrated in FIG. 1 and discussed above exhibits linear growth inresponse to initiator. However, increasing the rate of polymer growthcan enhance the ability to detect the presence of low copy numbertargets, such as a single target molecule in a large test volume. Forexample, monomers can be designed to undergo triggered self-assemblyinto branched structures exhibiting quadratic growth or dendriticstructures exhibiting exponential growth. The exponential growth islimited by the available space such that it decreases to cubicamplification as the volume around the initiator fills. However, ifchain reactions products are able to dissociate, exponential growth canbe maintained until the supply of monomers is exhausted.

In order to achieve non-linear growth, 3 or more HCR monomers can beused. In preferred embodiments at least 4 HCR monomers are used. In someembodiments, at least one monomer in a primary monomer pair incorporatea trigger nucleic acid segment that is complementary to the exposedsticky end of one of the monomers from a secondary set of HCR monomers.Upon exposure to the nucleic acid that is to be detected, the set ofprimary monomers undergoes HCR to form a polymer with a periodic singlestranded trigger region. Thus the trigger nucleic acid is exposed,leading to a polymerization chain reaction in the secondary set ofmonomers. In other embodiments, both the primary and secondary set ofmonomers includes a trigger segment, such that exponential growth isachieved. Exemplary schemes are presented in FIGS. 2 and 3 for achievingquadratic and exponential growth, respectively.

In one embodiment, one of a first pair of monomers comprises a bulgeloop. Upon polymerization, a single stranded region results from thepresence of the bulge loop. The bulge loop segment is preferablycomplementary to the sticky end of one of a second pair of HCR monomers.Thus, upon exposure to the initiator, the first pair of monomersundergoes HCR to form a polymer with a single stranded region that actsto trigger polymerization of the second pair of monomers. FIGS. 2A-Cdepict such a quadratic amplification scheme. Monomers Q1 and Q2interact with hairpin monomers H1 and H2 (FIG. 1) after initiation byI_(Q) to form the branched polymer schematically illustrated in FIG. 2D.

Q1 and Q2 (FIG. 2 a) are metastable in the absence of the initiatorI_(Q). I_(Q) binds to Q1 and a subsequent strand displacementinteraction exposes segments f, e* and b* as shown in FIG. 2B. Thissingle-stranded region contacts sticky end of Q2 and a subsequent branchmigration exposes segments c, b*, d* and e*. Segment d* then interactswith another copy of Q1 at sticky end d, causing the hairpin to open upsuch that e* can also hybridize. At the same time, the exposed c segmentinitiates a linear HCR reaction with hairpins H1 and H2 (not shown). Theresulting branched polymer has a main chain comprising alternating Q1and Q2 segments and H1/H2 side chains branching off at each Q2 segment.

In a further embodiment, exponential growth is achieved in response toan initiator by combining two or more pairs of monomers. For example,monomer pair Q1 and Q2 (FIG. 2) can be used in conjunction with monomersE1 and E2 (FIG. 3) to obtain exponential growth in response to aninitiator. In the presence of nucleic acid segment cb*, E1 and E2 form alinear chain that includes periodic single stranded d*e* regions. Bydesign, the initiator sequence for E1/E2 matches the periodic singlestranded region produced by Q1/Q2 and vice versa. Consequently, amixture of Q1, Q2, E1 and E2 monomers in the presence of initiator willform a structure in which each branch of the polymer is itself abranched polymer. Either initiator cb*, corresponding to the sticky endand first complementary region of E1, or d*e*, corresponding the stickyend and first complementary region of Q1, will activate the chainreaction.

While non-linear amplification systems provide enhanced sensitivity overa linear system, they may also have an increased chance for spuriousinitiation of HCR and a resultant increase in false-positive signals.Several methods may be used to decrease the possibility for initiationof the system in the absence of the initiator. In systems utilizinghairpin monomers, these may include helix clamping, helix elongation andloop entropy ratchets.

The quadratic and exponential growth HCR schemes illustrated in FIGS. 2and 3 include long single-stranded regions (b* and e* respectively).These long regions could potentially function as weak initiators.Several methods are available to reduce spurious monomer polymerizationin the absence of initiator for both higher order growth schemes andlinear growth schemes. These include helix clamping, helix lengtheningand loop entropy ratchets. In helix clamping, the single strandedregions in one or more of the monomers are truncated at each end so thatthe helixes that they could potentially invade in other monomers areeffectively clamped at the ends by bases that are not present in thesingle stranded (b* and e*) regions. Experiments have shown that thiscan eliminate any spurious initiation. The amount of truncation that iseffective to decrease or eliminate spurious initiation can be determinedby routine experimentation. For example, control experiments can beperformed using fluorescent gel electrophoresis time courses to monitorstrand exchange between single stranded DNA and duplex DNA (e.g., strandb* invading duplex bb*) for different clamp lengths. Using spectrallydistinct dyes for the initially single stranded DNA and for the two DNAspecies in the duplex allows independent monitoring of all species asstrand exchange proceeds. These controls can provide a systematic basisfor section of clamp dimensions.

The length of the helices in the linear HCR scheme illustrated in FIG. 1contributes directly to the height of the kinetic barrier that preventsspurious polymerization between the two hairpin species. Interactionsbetween H1 and H2 are sterically impeded by the loop size. However, thelong helices (bb*) in each hairpin provide a more fundamental kineticbarrier; the length of the helices has a direct effect on the height ofthe kinetic barrier that impedes spurious HCR. An increase in the lengthof the helices will increase the initial kinetic barrier in theuninitiated system. Thus, in some embodiments utilizing hairpinmonomers, for example if spurious initiation is observed, the length ofthe duplex region can be increased to reduce the background noise. Thehelix length necessary to reduce polymerization in the absence ofinitiator to an acceptable level can be readily determined by routineexperimentation. In some embodiments helix lengthening is combined withhelix clamping.

In still other embodiments utilizing hairpin monomers, loop entropyratchets are used to reduce HCR in the absence of initiator. Aninitiator opens an HCR hairpin via a three-way branch migration. Thisreaction is reversible because the displaced strand is tethered in theproximity of the new helix. However, by increasing the length of thesingle-stranded loop, the entropy penalty associated with closing theloop increases. As a result, a longer loop will bias the reaction toproceed forward rather than returning to the uninitiated state. However,larger loops are more susceptible to strand invasion. To counter thiseffect and allow the use of larger loops, mismatches can be introducedbetween the loop sequences and the complementary regions of the othermonomers. Again, the loop length and amount of mismatch that producesthe desired reduction in non-specific HCR can be determined by theskilled artisan through routine experimentation.

Initiator

The initiator is preferably a nucleic acid molecule. The initiatorcomprises an initiator region that is complementary to a portion of anHCR monomer, preferably a portion of the monomer that is available forhybridization with the initiator while the monomer is in its kineticallystable state. The initiator also preferably comprises a sequence that iscomplementary to a portion of the monomer adjacent to the sticky endsuch that hybridization of the initiator to the sticky end causes aconformational change in the monomer and begins the HCR chain reaction.For example, the initiator may comprise a region that is complementaryto the first complementary region of the HCR monomer, as describedabove.

In the preferred embodiments, the sequence of the initiator iscomplementary the sticky end (initiator complementary region) and firstcomplementary region of a first monomer. As described above, in someembodiments this will also influence the sequence of the secondcomplementary region and the loop of the second monomer species.

In some embodiments the initiator is a nucleic acid that is to bedetected in a sample or a portion of a nucleic acid that is to bedetected. In this case, the sequence of the target nucleic acid is takeninto consideration in designing the HCR monomers. For example, theinitiator complement region, preferably a sticky end, of one monomer isdesigned to be complementary to a portion of the target nucleic acidsequence. Similarly, a region adjacent to the sticky end of the samemonomer can be designed to be complementary to a second region of thetarget sequence as well. Because the second monomer will hybridize tothe first monomer, the sequence of the second monomer will also reflectat least a portion of the sequence of the target nucleic acid.

In other embodiments, the initiator comprises at least a portion of anucleic acid that is part of a “initiation trigger” such that theinitiator is made available when a predetermined physical event occurs.In the preferred embodiments that predetermined event is the presence ofan analyte of interest. However, in other embodiments the predeterminedevent may be any physical process that exposes the initiator. Forexample, and without limitation, the initiator may be exposed as aresult of a change in temperature, pH, the magnetic field, orconductivity. In each of these embodiments the initiator is preferablyassociated with a molecule that is responsive to the physical process.Thus, the initiator and the associated molecule together form theinitiation trigger. For example, the initiator may be associated with amolecule that undergoes a conformational change in response to thephysical process. The conformational change would expose the initiatorand thereby stimulate polymerization of the HCR monomers. In otherembodiments, however, the initiation trigger comprises a single nucleicacid. The initiator region of the nucleic acid is made available inresponse to a physical change. For example, the conformation of theinitiation trigger may change in response to pH to expose the initiatorregion.

The structure of the trigger is preferably such that when the analyte ofinterest is not present (or the other physical event has not occurred),the initiator is not available to hybridize with the sticky end of amonomer. Analyte frees the initiator such that it can interact with ametastable monomer, triggering the HCR polymerization reactionsdescribed above. In some embodiments analyte causes a conformationalchange in the trigger that allows the initiator to interact with themonomer.

The initiator may be part of a trigger comprising a nucleic acid that islinked to or associated with a recognition molecule, such as an aptamer,that is capable of interacting with an analyte of interest. The triggeris designed such that when the analyte of interest interacts with therecognition molecule, the initiator is able to stimulate HCR.Preferably, the recognition molecule is one that is capable of bindingthe analyte of interest.

Recognition molecules include, without limitation, polypeptides, such asantibodies and antibody fragments, nucleic acids, such as aptamers, andsmall molecules. The use of an initiator bound to an aptamer isdescribed in more detail below.

In some particular embodiments, amplification of diverse recognitionevents is achieved by coupling HCR to nucleic acid aptamer triggers. Anaptamer is identified that is able to specifically bind an analyte ofinterest. The analyte is not limited to a nucleic acid but may be, forexample, a polypeptide or small molecule. The aptamer is linked to anucleic acid comprising an initiator region in such a way that theinitiator is unavailable to stimulate HCR in the absence of analytebinding to the aptamer.

Preferably, conformational changes in the aptamer secondary structureexpose the initiator segment. In one embodiment, such an aptamer triggeris a hairpin nucleic acid that comprises an initiator segment that iscomplementary to the initiator complement region or sticky end of an HCRmonomer. The aptamer trigger also comprises a complementary region thatis complementary to a region of the HCR monomer adjacent to the stickyend, a loop region and an aptamer sequence. The hairpin aptamer triggermay also comprise a region that enhances the stability of the hairpin inthe absence of aptamer binding to the analyte, such as a nucleic acidregion in one arm of the hairpin that is complementary to a region ofthe other arm.

FIG. 5A depicts a scheme for HCR amplification of ATP binding using anaptamer construct that exposes an initiator strand upon ATP binding. Thesticky end can act as a trigger for the HCR mechanism of FIG. 1 byopening hairpin H2. The region x is introduced to help stabilize thetrigger in the absence of analyte. The region b* includes both thehairpin loop and the portion of the stem complementary to x. Thistrigger mechanism is based on conformational changes in the aptamersecondary structure (Yingfu Li (2003) Journal of the American ChemicalSociety 125:4771-4778) that make the initiator strand available tostimulate HCR. FIG. 5B illustrates successful detection of ATP, as wellas specificity in differentiating ATP from GTP, as discussed in moredetail in the Examples below.

Detecting HCR

The products of HCR are readily detectable by methods known to one ofskill in the art for the detection of nucleic acids, including, forexample, agarose gel electrophoresis, polyacrylamide gelelectrophoresis, capillary electrophoresis, and gel-filled capillaryelectrophoresis. As the polymers comprise nucleic acids, they can bevisualized by standard techniques, such as staining with ethidiumbromide. Other methods also may be suitable including light scatteringspectroscopy, such as dynamic light scattering (DLS), viscositymeasurement, colorimetric systems and fluroscence spectropscopy. Asdiscussed in more detail below, in the preferred methods for in situimaging and detection, HCR products are fluorescently labeled.

In some embodiments HCR is monitored by fluorescence resonance energytransfer (FRET). Certain monomers are labeled with fluorescent dyes sothat conformational changes resulting from HCR can be monitored bydetecting changes in fluorescence. In one embodiment, one of a pair ofhairpin molecules is labeled with a fluorophore at the junction of theregion complementary to the initiator strand and the duplex region andlabeled at the opposing side of the duplex region with a quenchermolecule. Upon polymerization, the fluorophore and quencher areseparated spatially in the aggregated nucleic acid structure, providingrelief of the fluorescence quenching. In this case, the presence of asingle initiator is amplified by the chain of fluorescent events causedby HCR. In the context of in situ imaging, the presence of a singletarget molecule can be amplified by the chain of fluorescence events. Inaddition, for in situ imaging the quenching of fluorescence in unreactedmonomers reduces background noise. Thus, unreacted monomers do not needto be removed from the sample.

Because the size of the HCR products is inversely related to the amountof the target analyte in a sample, HCR can be used to determine analyteconcentration. The average molecular weight of the HCR products isobtained by standard measurements. Is some embodiments the averagemolecular weight of HCR products obtained from one sample is compared tothe average molecular weight of HCR products from one or more othersamples with an unknown analyte concentration. In this way, the relativeconcentration of analyte in each sample can be determined.

In other embodiments, the concentration of analyte is determined bycomparing the average molecular weight from a sample with unknownconcentration to the average molecular weight of HCR products from HCRreactions in one or more control samples with a known concentration ofthe analyte. In this way the concentration of analyte in the unknownsamples can be determined to be the same as one of the control samples,greater than one of the control samples, less than one of the controlsamples, or in the rance of concentration between two control samples.Thus, the number of control reactions can be adjusted based on theparticular circumstances to provide more or less sensitive determinationof analyte concentration. For example, if a relatively exact analyteconcentration is necessary, the number of control samples can beincreased. On the other hand, if only a broad idea of analyteconcentration is necessary, fewer control samples can be used.

Application to In Situ Imaging

HCR provides an enzyme-free approach to in situ amplification that canbe multiplexed in parallel. Furthermore, HCR amplification provides ameans for reducing the background signal resulting from nonspecificprobe binding. Probes for the target to be detected in a biologicalsample incorporate an HCR initiator. After binding to the target, theinitiator triggers the self-assembly of tethered (to the target)non-covalent ‘polymers’ built from HCR monomers, preferably hairpinmonomers as described above. The HCR monomers are preferablyfluorescently labeled so that the polymers can be detected and thepresence and/or location of the target determined.

In addition to an initiator, probes also preferably comprise a targetregion that is able to specifically bind to or associate with a target.In some embodiments, particularly where the target is a nucleic acid,such as a gene or mRNA, the probe target region comprises a nucleicacid. In other embodiments the probe target region comprises apolypeptide, such as an antibody (FIG. 9).

The HCR monomers are also referred to herein as “amplifiers” because thepolymerization of the monomers upon binding of a probe to the targetproduces a detectable signal, which is amplified compared to the signalthat would be produced by the binding of a single probe to the target.The amplifiers can each be labeled with the same or differentfluorophores. For example, the system can be designed to use more thantwo monomer species per target, with at least one species fluorescentlylabeled. Fluorescent labels are well known to one of skill in the artand include those, for example, in the “The Handbook—A Guide toFluorescent Probes and Labeling Technologies,” 10th Edition (availableat http://www.probes.com).

In some embodiments, amplifiers within the system are labeled with botha fluorophore and a quencher to form a construct analogous to a“molecular beacon” (Tyagi et al. Nature Biotechnology 14:303-308, 1996).For example, a hairpin monomer can comprise both a fluorophore and aquencher, such that the quencher reduces fluorescence while the monomeris in the hairpin form but not when the monomer is incorporated into anHCR polymer. Thus, molecular beacon versions of HCR monomers can reducebackground signal resulting from any unpolymerized monomers, such asthose that bind non-specifically or that simply remain unreacted in thesample.

For imaging of biological samples, it is advantageous to use amplifiercomponents that are small in size to allow penetration into the sample.For example, in some embodiments HCR components less than about 20nanometers are used. More preferably the HCR components are less thanabout 15 nm, even more preferably less than about 10 nm, and still morepreferably between about 8 and 16 nm. In some particular embodiments theHCR monomers are less than about 8 nm. Standard procedures for in situimaging can be used to cause the HCR products to enter the sample. Theskilled artisan will be able to select the appropriate methods forcausing the HCR components to enter the sample.

Active Background Suppression Using Triggered Probes and Self-QuenchingComponents

For in situ hybridization studies, the simplest probe designincorporates an initiator strand at the end of the probe molecule (FIG.8A). The probe portion of the probe is specific for the target moleculethat is to be detected in the sample. For example, the probe portion cancomprise a nucleic acid that is complementary to a portion of a gene ofinterest or to an mRNA of interest. In another embodiment the probecomprises a protein or nucleic acid, such as an aptamer, that is able tobind to or associate with the target. For example, the probe maycomprise an antibody, antibody fragment, DNA binding protein or othernucleic acid or polypeptide that is able to bind a target of interest.In one embodiment, illustrated in FIG. 9, the probe comprises anantibody that is specific for a protein of interest coupled to aninitiator.

After performing probe hybridization (or binding) and washing the sampleto remove unbound probes, HCR hairpins can be introduced. The presenceof the initiator portion of the probe causes the HCR hairpins (oramplifiers) to self-assemble “polymers,” essentially as described abovefor general HCR. However, in this context, the polymers will remaintethered to the target , such as an mRNA. In some preferred embodimentsthe amplifiers are fluorescently labeled, such that the resultingpolymers have about 8 nm dye spacing.

If the probe of FIG. 8A binds nonspecifically in the sample, then theinitiator will cause HCR amplification at a location where no target wasdetected. All fluorescent in situ hybridization methods currently sufferfrom the problem of background signal resulting from nonspecific probebinding. However, with HCR amplification it is possible to avoidexcessive non-specific background with the use of a triggered probe.

Using HCR amplification, it is conceptually straightforward to design“triggered probes” that actively suppress background from nonspecificbinding, particularly when the target molecule is a nucleic acid. Onesuch probe is illustrated in FIG. 8B. With a triggered probe, specificprobe binding to a nucleic acid target exposes the initiator strand viaa strand displacement interaction. The initiator is then kineticallyimpeded from returning to its protected state by the base pairs formedbetween the target and the previously looped portion of the probe (thestrength of this free energy ratchet can be adjusted by changing thesize of the loop). However, if nonspecific probe binding occurs, theinitiator remains protected and HCR amplification is prevented.

The triggered probe design of FIG. 8B implies that subsequences a and bof the HCR amplifier hairpins are constrained to be within a window ofconsecutive bases from a target mRNA (a=6 bases, b=18 bases for thestandard HCR hairpin design). If desirable, the dependence of theamplifier hairpins on the target sequence can be reduced using thedesign of FIG. 8C, in which only subsequence “a” is determined by thetarget mRNA sequence. This change dramatically increases the flexibilityin designing multiple orthogonal amplifier systems. If it becomesdesirable to eliminate the sequence dependence completely, this can alsobe accomplished using an additional branch migration step (not shown).

In other embodiments, the triggered probe is configured such that achange in conformation upon target binding releases an initiator strandor otherwise makes the initiator region available to interact with anHCR monomer to initiate polymerization. Such probes can be used, forexample, to detect nucleic acids, but also to detect targets that arenot nucleic acids, such as polypeptides.

Once a triggered probe has been activated by binding (or associating)specifically to a target, fluorescently labeled HCR monomers, preferablyhairpins, can self-assemble into a tethered amplification polymer.Because the initiator is protected prior to target binding, it is notnecessary to remove unbound probe prior to providing HCR monomers to thesystem. Thus, HCR monomers can be provided to the system at the sametime as the triggered probe. However, in some embodiments unbound probeis washed prior to providing the HCR monomers.

FIGS. 10A and 10C illustrate standard end-labeled hairpins (FIG. 10A)and the corresponding amplification polymer (FIG. 10C) with dyes placedat about 8 nm spacing. For some samples, such as fixed whole mountsamples, unused labeled monomers can be washed from the sample to reducebackground signal. However, this approach is not suitable for allsamples and all applications. For example, it is not viable for in vivoimaging. Thus, in some embodiments active background suppression caninstead be achieved by making the HCR hairpins self-quenching usingfluorophore/quencher pairs (FIGS. 10B). In the hairpin form (FIG. 10B),fluorophore/quencher pairs have about 2 nm spacing, but the hairpins arelabeled so that each fluorophore is about 8 nm from the nearestquenchers after amplification occurs (FIG. 10D). After polymerization,the neighboring dyes will have about 2 nm separation. Within a givenpolymer, the dyes can be chosen to be identical (a single-coloramplifier) or to have non-overlapping excitation and emissions spectra.

Quantitative Amplification Via Triggered Self-Assembly of Fixed-SizeAmplification Polymers

To increase the sensitivity of HCR schemes while achieving a fixed levelof amplification per target molecule to provide quantitative signalstrength, branched nonlinear amplification schemes that form polymers ofa pre-defined size can be used (FIG. 11). Amplification components areshown in FIG. 11A. The amplification polymer branches to form a binarytree (FIG. 11B) using two HCR monomer species for each generation ofbranching (FIG. 11C).

In the absence of steric effects, the polymer will grow exponentially toa prescribed size determined by the number of hairpin generations thatare in solution. If each hairpin is labeled with F fluorophores, a treewith G generations requires 2G−1 hairpins and yields F(2^(G−)1) dyestethered to the triggered probe. Only the first two generations(comprising hairpins H1, H2A, and H2B) are related to the probesequence, so subsequent generations can be used as standardizedamplifier components. Hence, after designing and synthesizing componentsfor M orthogonal amplification trees, the components can be re-used forsimultaneous multiplexing of any M targets of interest. For activebackground suppression in vivo, self-quenching hairpins can be obtainedby again using fluorophore/quencher pairs.

In an analogous fashion to the self-quenching scheme of FIGS. 10B and10D, the labels can be arranged so that quenchers are clustered awayfrom the fluorophores in the assembled polymer. After some number ofgenerations, exponential growth must slow to cubic growth as the volumearound the initiator fills. To increase the number of generations ofexponential growth, spacer regions can be introduced into the hairpinsto increase the separation between clusters of the same generation.

Imaging Multiple Analytes

A major conceptual benefit of HCR amplification is the ability toamplify multiple targets simultaneously. HCR targeting a number ofanalytes (for example, gene transcripts or proteins) can be usedsimultaneously. In one embodiment, each HCR system is labeled with aspectrally distinguishable dye. Accordingly, the number of analytes isequal to the number of spectrally distinguishable dyes that areavailable. For many situations, this will be sufficient.

To study the expression of multiple mRNAs or proteins, it is desirableto perform multiplexed amplification of all recognition eventssimultaneously using orthogonal HCR amplifiers. To maximize the numberof distinct targets that can be imaged using a limited supply ofspectrally distinct fluorophores, the unamplified combinatorialmultiplexing approach of Levsky and co-workers (Levsky et al. Science297:836-840, 2002) can be adapted for HCR amplification by labeling themonomers for each amplifier with different unique dye combinations Theuse of barcodes with a minimum of two colors provides a basis forscreening single-color signals resulting from probes that are not boundspecifically. However, triggered probes for HCR amplification alreadyhave a built-in capability to reduce background.

Therefore, in some embodiments only a single probe is used for eachtarget and combinatorial multiplexing is performed by labeling themonomers for each HCR amplifier with different unique dye combinations.This approach is preferable to a combinatorial approach in which youjust make a probe for a target that you then independently amplify withtwo one-color systems. Firstly, that method doesn't work forsingle-molecule detection, and second, there is no guarantee on therelative ratio of each dye that would be deposited in that case.

If the H1 and H2 hairpins are end-labeled with different dyes, the HCRproduct will carry an equal number of each dye by construction. Ingeneral, N spectrally distinct fluorophores can be used to address T:N!/[(N−2)!2!] targets with dual-color amplifiers (e.g., 4 dyes for 6targets, 5 dyes for 10 targets). However, since combinatorial barcodesare not employed as a background diagnostic using this approach, thenumber of targets can be increased to T=N! [N−2]!2!+N by allowingsingle-color amplifiers (e.g., 4 dyes for 10 targets, 5 dyes for 15targets). Furthermore, it is possible to label HCR monomers with morethan one dye to increase the number of targets that can be addressed upto T=Σ_((i−1,N))N!/[(N−i)!i!]=2^(N)−1 (e.g., 4 dyes for 15 targets, 5dyes for 31 targets). HCR systems also can be designed that used Mhairpins per amplifier.

As a proof of principle, FIG. 7 demonstrates simultaneous and specificdetection of two different DNA fragments using two HCR amplificationsystems.

EXAMPLES

HCR monomers comprising DNA sequences were designed using a combinationof criteria (Dirks et al. Nucleic Acids Research 32:1392-1403 (2004)).These included sequence symmetry minimization (Seeman N. C. J. Theor.Biol. 99:237-247 (1982)), the probability of adopting the targetsecondary structure at equilibrium (Hofacker et al. Monatsh. Chem.125:167-188 (1994)), the average number of incorrect nucleotides atequilibrium relative to the target structure (Dirks et al. Nucleic AcidsResearch 32:1392-1403 (2004)) and hybridization kinetics (Flamm et al.RNA 6:325-338 (2000)). The sequences of the monomers and initiator forthe basic HCR system illustrated in FIG. 1 and the aptamer trigger HCRsystem illustrated in FIG. 5 are shown in Table 1. The aptamer systemincluded new sequences to ensure compatibility with the fixed sequenceof the aptamer. DNA was synthesized and purified by Integrated DNATechnologies (Coralville, Iowa).

TABLE 1 System Strand Sequence* Basic H15′-TTA ACC CAC GCC GAA TCC TAG ACT CAA AGT AGT CTA GGA TTC GGC GTG-3′(SEQ ID NO: 1) H2 5′-AGT CTA GGA TTC GGC GTG GGT TAACAC GCC GAA TCC TAG ACT ACT TTG-3′ (SEQ ID NO: 2) I5′-AGT CTA GGA TTC GGC GTG GGT TAA-3′ (SEQ ID NO: 3) Aptamer^(†) H15′-CAT CTC GGT TTG GCT TTC TTG TTA CCC AGG TAA CAA GAA AGC CAA ACC-3′(SEQ ID NO: 4) H2 5′-TAA CAA GAA AGC CAA ACC GAG ATGGGT TTG GCT TTC TTG TTA CCT GGG-3′ (SEQ ID NO: 5) I^(ATP)5′-CCC AGG TAA CAA GAA AGC CAA ACC TCT TGT 

 

 

 

 

 

 

 

 

 

-3′ (SEQ ID NO: 6) I 5′-CCC AGG TAA CAA GAA AGC CAA ACC-3′(SEQ ID NO: 7) *In the hairpin sequences, loops are in bold and stickyends are italicized. ^(†)Aptamer nucleotides are italicized and bolded.

For the basic HCR system illustrated in FIG. 1, concentrated DNA stocksolutions were prepared in buffer that was later diluted to reactionconditions. The buffer comprised 50 mM Na2HPO4/0.5M NaCl (pH 6.8).

Monomers H1 and H2 (FIG. 1B) were mixed at various concentrations in thepresence and absence of initiator. Stock solutions of I, H1 and H2 werediluted in reaction buffer to three times their final concentration and9 μl of each species was combined, in that order to give a 27 μlreaction volume. Six different concentrations of initiator were used(0.00, 10.00, 3.20, 1.00, 0.32 and 0.10 μM) in a 1 μM mixture of H1 andH2. Reactions were incubated at room temperature for 24 hours beforerunning 24 μl of each product on a gel. Samples were loaded on 1%agarose gels containing 0.5 μg EtBr per ml of gel volume. The gels wereprepared using 1× SB buffer (10 mM NaOH, pH adjusted to 8.5 with boricacid). The agarose gels were run at 150 V for 60 minutes and visualizedunder UV light.

FIG. 1D illustrates the results of the HCR reactions and the effect ofinitiator concentration on amplification. Lanes 2-7 of the gel shown inFIG. 1D are the results of the HCR reactions at the various initiatorconcentrations, respectively. Lanes 1 and 8 are DNA markers with 100-bpand 500-bp increments respectively. As illustrated by FIG. 1D,introduction of an initiator strand triggers a chain reaction ofhybridization that results in the production of polymers of varioussizes. The average molecular weight of the polymers is inversely relatedto the initiator concentration (FIG. 1( d)). The inverse relationshipfollows from the fixed supply of monomer hairpins, but the phenomenonwas observed after 10 minutes, when the supply of monomers had not beenexhausted.

These results confirmed an earlier experiment in which 1 μM of H1 and H2were reacted overnight in 0.5M NaCl, 50mM Na₂HPO₄ at pH 6.5 withinitiator at concentrations of 0, 1, 0.1, 0.01, 0.001 and 0.0001 μM.With no initiator HCR reactions were not observed. In addition, novisible polymer growth was observed at initiator concentrations of 0.001and 0.0001 μM. At the other initiator concentrations an inverserelationship was observed between the initiator concentration and theaverage molecular weight of the resulting polymers.

In another set of experiments, the aptamer trigger illustrated in FIG.5A was utilized. The aptamer trigger (I^(ATP)) comprised an initiatorregion corresponding to the initiator used in the experiments describedabove, linked to an aptamer that is able to specifically interact withATP (Huizenga et al. Biochemistry 34:656-665 (1995)). In addition, theaptamer trigger comprises a stabilizing region designed to stabilize thetrigger in the absence of ATP. The aptamer trigger is designed such thatin the presence of ATP the hairpin is opened and the initiator regionexposed, thereby triggering polymerization of the H1 and H2 monomers.The sequence of I^(ATP) is provided in Table 1, above.

Reactions were carried out with various combinations of H1, H2, I,I^(ATP), ATP and GTP. Concentrated stock solutions of the constituentswere diluted to reaction conditions: 5 mM MgCl2/0.3 M NaCl/20 mM Tris(pH 7.6). Reactions were performed with 1.4 mM ATP and/or GTP. DNAspecies were combined to yield 1 μM concentrations in 27 μl of reactionbuffer, with additions made in the following order: buffer and/orinitiator I or aptamer trigger I^(ATP), H1, and then H2 (I and I^(ATP)interact with H2 rather than H1). In this case, 1 μl of 40 mM ATP, 40 mMGTP or water was added to each reaction, as appropriate, for a totalreaction volume of 28 μl.

Reactions were incubated at room temperature for one hour and run onagarose gels (as described above) or native polyacrylamide gels. Nativepolyacrylamide gels were 10% precast gels made with 1× TBE buffer (90 mMTris, 89 mM boric acid, 2.0 mM EDTA, pH 8.0). The polyacrylamide gelswere run at 150V for 40 minutes in 1× TBE and stained for 30 minutes ina solution containing 5 μg EtBr per ml.

FIG. 5B shows a representative agarose gel illustrating that the aptamertrigger I^(ATP) can initiate polymerization of H1 and H2 in the presenceof ATP and that ATP can be distinguished from GTP. Hairpins H1 and H2did not polymerize when mixed in the absence of initiator (H1+H2; Lane1), but did polymerize when the initiator I was added (H1+H2+I; Lane 2).ATP alone was unable to trigger the polymerization of the hairpinmonomers (H1+H2+ATP; Lane 3) and no polymers were observed from thecombination of aptamer trigger with ATP in the absence of hairpinmonomers (IATP+ATP; Lane 4). Some weak spurious HCR was observed in theabsence of ATP from the combination of monomers and aptamer trigger(H1+H2+I^(ATP); Lanes 5) or in the presence of GTP (H1+H2+I^(ATP)+GTP;Lane 7), respectively. Strong HCR amplification of ATP recognition wasseen when the monomers were combined with the aptamer trigger in thepresence of ATP (H1+H2+I^(ATP)+ATP; Lane 6). A DNA ladder is shown inLane 8 (100-1000 by in 100 by increments).

The kinetics of HCR reactions were explored using fluorescencequenching. The adenine analog 2-aminopurine (2AP) fluoresces when singlestranded but is significantly quenched when in a stacked double-helicalconformation (Rachofsky et al. Biochemistry 40: 996-956 (2001)). Monomerusage was monitored as polymerization occurred by replacing H1 with thelabeled hairpin H1^(2AP). H1^(2AP) was prepared by substituting 2AP forthe third base (A) in the sticky end of H1 (see Table 1). Monitoring 2APfluorescence was used rather than standard end-labeled strands becausethe local environment of quenched 2AP was the same regardless of whetherinitiator (I) or monomer (H2) performs the quenching.

Fluorescence data were obtained using a fluorometer from PhotonTechnology International (Lawrenceville, NJ), with the temperaturecontroller set to 22° C. Excitation and emission wavelengths were 303and 365 nm, respectively, with 4 nm bandwidths. Stock solutions of 0.40μM H12AP and 0.48 μM H2 were prepared in reaction buffer as describedabove, heated to 90° C. for 90 seconds and allowed to cool to roomtemperature for 1 hour before use. For each experiment, 250 μl of H12APwas added to either 250 μl of H2 or 250 μl of reaction buffer. These0.20 μM H12AP solutions were allowed to sit at room temperature for atleast 24 hours before fluorescence measurements were taken. The initialsignal was obtained after rapidly pipetting the sample in the cuvette toobtain a stable fluorescence baseline. After acquiring at least 2,000seconds of the baseline, runs were paused for about 1 minute to add 20μl of initiator (either 20 μM or 2.5 μM) and allow mixing by rapidpipetting. The final reaction volume was 520 μl for all experiments. Thevariation in initial fluorescence intensities was about 10% across threeexperiments.

As evidenced by the results presented in FIG. 1E the hairpin monomers H1and H2 do not hybridize in the absence of initiator. Addition ofinitiator (I) to the hairpin mixture led to fluorescence quenching viaHCR (bottom band from 0 to 2000 seconds, then dropping to middle bandfrom 2000 seconds on)

The same quenched baseline was achieved without HCR by combiningH1^(2AP) with excess initiator in the absence of H2 (FIG. 1(E), middleband from 0 to 2000 seconds, then dropping to bottom band from 2000seconds on). In this case, each initiator molecule caused onefluorescent signaling event by binding to H1^(2AP). With H2 present, HCRperformed fluorescent amplification, allowing each initiator molecule toalter the fluorescence of multiple hairpins.

Addition of insufficient initiator to H1^(2AP) provided only partialquenching (FIG. 1(E), top band), demonstrating that HCR, and notinitiator alone, was responsible for exhausting the supply of H1^(2AP)monomer.

Although the foregoing invention has been described in terms of certainpreferred embodiments, other embodiments will be apparent to those ofordinary skill in the art. Additionally, other combinations, omissions,substitutions and modification will be apparent to the skilled artisan,in view of the disclosure herein. Accordingly, the present invention isnot intended to be limited by the recitation of the preferredembodiments, but is instead to be defined by reference to the appendedclaims. All references cited herein are incorporated by reference intheir entirety.

1-27. (canceled)
 28. A system for forming a structure comprisinghybridized nucleic acid monomers comprising: a nucleic acid initiatorcomprising a first region, a second region and a third region; a firstnucleic acid hairpin monomer comprising: a first region comprising asticky end, wherein the sticky end is located at an end of the monomerand is capable of hybridizing to the first region of the initiator; asecond region adjacent to the first region, wherein the second region iscapable of hybridizing to the second region of the initiator; a thirdregion adjacent to the second region, wherein the third region iscapable of hybridizing to the third region of the initiator; a fourthregion that is hybridized to the third region of the first monomer whenthe initiator is not hybridized to the first monomer; and a fifth regionthat is hybridized to the second region of the first monomer when theinitiator is not hybridized to the first monomer; and a second hairpinmonomer comprising: a first region comprising a sticky end located at anend of the monomer, wherein the sticky end is capable of hybridizing tothe fourth region of the first monomer.
 29. The system of claim 28,wherein the second and third regions and fourth and fifth regions of thefirst monomer form a duplex stem when the initiator is not hybridized tothe first monomer.
 30. The system of claim 28, wherein the fourth regionof the first hairpin monomer hybridizes to the sticky end of the firstregion of the second hairpin monomer if the first, second and thirdregions of the first hairpin monomer hybridize to the initiatormolecule.
 31. The system of claim 28, wherein the first hairpin monomerfurther comprises a sixth region and a seventh region that are capableof hybridizing to the third region and second region of the secondhairpin monomer, respectively.
 32. The system of claim 31, wherein saidfourth region of the first hairpin monomer initiates hybridization ofsaid sixth region and seventh regions of the first hairpin monomer tosaid third region and second region of said second hairpin monomer,respectively, if the first, second and third regions of the firstmonomer hybridize to the first, second and third regions of theinitiator.
 33. The system of claim 30, wherein said sixth and seventhregions of the first hairpin monomer comprises a portion of a singlestranded hairpin loop.
 34. The system of claim 28, wherein the firsthairpin monomer further comprises an eighth region and a ninth regionthat are single stranded and located at an end of the monomer oppositethe sticky end of the first region.
 35. The system of claim 28,additionally comprising a third hairpin monomer comprising: a firstregion comprising a sticky end located at an end of the third hairpinmonomer, wherein the sticky end is capable of hybridizing to the fifthregion of the first hairpin monomer.
 36. The system of claim 35, whereinthe third hairpin monomer additionally comprises a second region and athird region that are capable of hybridizing to the eighth region andninth region of the first hairpin monomer.
 37. A method for forming astructure comprising hybridized nucleic acid monomers, the methodcomprising: providing a first hairpin monomer comprising: a first domaincomprising a first sticky end, wherein the first sticky end is availableto hybridize to an initiator that comprises a nucleic acid sequencecomplementary to the first domain; a single stranded loop; and a seconddomain, wherein a portion of the second domain is complementary to aportion of the first domain; providing a second hairpin monomercomprising: a first domain comprising a first sticky end, wherein thefirst sticky end comprises a nucleic acid sequence complementary to aportion of the second domain of the first monomer; a single strandedloop; and a second domain, wherein a portion of the second domain ishybridized to a portion of the first domain; and combining the first andsecond monomers with the initiator, wherein the initiator hybridizes tothe first domain of the first monomer, and subsequently the first domainof the second monomer hybridizes to the second domain of the firstmonomer, thereby forming a structure comprising the first and secondmonomers.
 38. The method of claim 37, wherein the second domain of thefirst hairpin monomer comprises a portion of the single stranded loop.39. The method of claim 37, wherein the first hairpin monomer furthercomprises a third domain, wherein a portion of the third domain ishybridized to a portion of the first domain prior to hybridization ofthe initiator to the first domain of the first monomer.
 40. The methodof claim 39, wherein the third domain of the first hairpin monomer iscapable of hybridizing to a first domain of a third nucleic acid hairpinmonomer.
 41. The method of claim 39, wherein the first domain of thethird hairpin monomer comprises a first sticky end that is capable ofhybridizing to the portion of the third domain of the first hairpinmonomer that is hybridized to the portion of the first domain prior tohybridization of the initiator to the first domain of the first monomer.42. A kit comprising: a first nucleic acid hairpin monomer comprising: afirst section having a first nucleic acid sequence; a second sectionhaving a second nucleic acid sequence; a third section having a thirdnucleic acid sequence; a fourth section having a fourth nucleic acidsequence; a fifth section having a fifth nucleic acid sequence; a sixthsection having a sequence complementary to the third nucleic acidsequence; a seventh section having a sequence complementary to thesecond nucleic acid sequence; an eighth section having the fourthnucleic acid sequence; and a ninth section having the fifth nucleic acidsequence; and a second nucleic acid hairpin monomer comprising: a firstsection having the third nucleic acid sequence; a second section havinga sequence complementary to the fifth nucleic acid sequence; a thirdsection having a sequence complementary to the fourth nucleic acidsequence; a fourth section having a sixth nucleic acid sequence; a fifthsection having a seventh nucleic acid sequence; a sixth section havingthe fourth nucleic acid sequence; a seventh section having the fifthnucleic acid sequence; an eighth section having the sixth nucleic acidsequence; and a ninth section having the seventh nucleic acid sequence.43. The kit of claim 42, additionally comprising: a third nucleic acidhairpin monomer comprising: a first section having the second nucleicacid sequence; a second section having a sequence complementary to thefourth nucleic acid sequence; a third section having a sequencecomplementary to the fifth nucleic acid sequence; a fourth sectionhaving the seventh nucleic acid sequence; a fifth section having thesixth nucleic acid sequence; a sixth section having the fifth nucleicacid sequence; a seventh section having the fourth nucleic acidsequence; an eighth section having the seventh nucleic acid sequence;and a ninth section having the sixth nucleic acid sequence.